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RESEARCH COMMUNICATION |
a ,b
a Laboratory of Molecular Signalling, Babraham Institute, Cambridge, England, U.K.;
b Laboratory of Biocybernetics, Faculty of Electrical Engineering, University of Ljubljana, Slovenia; and
c Department of Zoology, University of Cambridge, Cambridge, England, U.K.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, F., Bootman, M. D., Berridge, M. J., Parkinson, N. A., Lipp, P. Elementary
[Ca2+]i signals generated by electroporation
functionally mimic those evoked by hormonal stimulation.
Key Words: Ca2+ puffs • Ca2+ waves • HeLa cells • confocal microscopy • InsP3 receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously observed that Ca2+ puffs both initiated and propagated Ca2+ waves in hormonally stimulated HeLa cells (4) . The Ca2+ puffs evoked during agonist stimulation of HeLa cells are highly localized Ca2+ signals (~6 µM in diameter), arising from clusters of InsP3 receptors (InsP3Rs) spaced ~6 µm apart (4) . They occur stochastically during cell stimulation, and unless adjacent elementary Ca2+ release sites become functionally coupled their Ca2+ signals remain spatially confined (12) . Essentially, each Ca2+ puff contributes a small quantum (a few attomoles) of Ca2+ into the cytoplasm. When the Ca2+ puff sites operate at low frequencies or amplitudes, the released Ca2+ can be effectively buffered or resequestered by the cell. However, with sufficient activity, the released Ca2+ can overwhelm the cellular buffering mechanisms, leading to a progressive [Ca2+]i increase that eventually triggers a regenerative Ca2+ wave (12 , 13 ).
In the present study, we used an electroporation device to evoke spatially restricted, rapid and reversible influx of Ca2+ into HeLa cells. Electroporation essentially involves the application of a high-intensity electric field across a cell, leading to changes in plasma membrane potential that cause a localized breakdown of the lipid bilayer 14-16) . The electroporation effect is a localized phenomenon because not all parts of the membrane exposed to an electrical field are subject to the same change in membrane potential 17-20) . In fact, the change in membrane potential, and consequently the electroporative effect, is most prominent at the restricted parts of the cell facing the electrodes 21-23) , particularly the membrane facing the anode, where the membrane potential becomes hyperpolarized. The extent and duration of permeabilization can be modulated by changing parameters such as strength, duration, and repetition frequency of the electric field. The use of electroporation to evoke spatially discrete [Ca2+]i changes allowed us to test the hypothesis that the transition from local to global [Ca2+]i signals is dependent on the amplitude and frequency of elementary Ca2+ signals (13) .
Electroporation of HeLa cells using low field intensities evoked Ca2+ puffs with the same spatio-temporal characteristics as hormone-evoked elementary events. These electroporation-induced signals were a consequence of the minute influx of Ca2+ that occurred during the rapid and transient plasma membrane disruption. Electroporation could functionally mimic the effect of a hormone on the cells in that it could trigger localized Ca2+ puffs or globally propagating regenerative Ca2+ waves.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For video imaging studies, a coverslip of fura-2-loaded cells was mounted in a homemade electropermeabilization chamber (see below). The chamber was placed on the stage of a Nikon Diaphot 300 inverted microscope. Video images were obtained using an intensified CCD camera (COHU, San Diego, Calif.), stepping filter wheel (Rainbow; Life Sciences Resources Ltd., U.K.), and Merlin acquisition system (Life Science Resources Ltd., Cambridge, U.K.). The cells were alternately excited at 340 nm and 380 nm using a 75W Xenon lamp in conjunction with 7.5 HBW filters (Omega Optical, Dallas, Tex.); fluorescence emission was measured at 510 nm (1 image per second). [Ca2+]i was estimated from the 340 nm/380 nm fluorescence ratio according to the equation: [Ca2+]i = Kd x ß ((R-Rmin)/(Rmax-R)) (25) . Rmin and Rmax were determined empirically by permeabilizing the cells to Ca2+ using 5 µM ionomycin (Sigma, Poole, Dorset, U.K.) and exposing the cells to extracellular solutions of high (10 mM) and low Ca2+ (no added CaCl2 +10 mM EGTA). Typical values obtained were Rmin = 0.15, Rmax = 2. The dissociation constant, Kd, was taken to be 225 nM (25) . Experiments investigating Mn2+ quench of fura-2 were performed using standard EM supplemented with 250 µM MnCl2. The fluorescent indicator was excited in Ca2+-insensitive absorption wavelength 360 nm (isosbestic point).
Confocal imaging
Confocal images were obtained using a Noran Oz laser-scanning
confocal microscope (Noran, Milton Keynes, U.K.). Fluo-3-loaded cells
mounted in the electroporation chamber were placed on the stage of a
Nikon Diaphot 300 microscope. Fluo-3 was excited using the 488 nm line
of an argon-ion laser, and the fluorescence emission was collected at
wavelengths >505 nm. The confocal slit was chosen to give a `z'
resolution of ~1 µm. Images were acquired with a rate of 7.5 images
per second. Data processing was performed as described previously
(4)
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Measurements of relative membrane potential changes were performed using di-8-ANEPPS-loaded cells as described by Hüser et al. (26) . The cells were loaded with di-8-ANEPPS by incubation with a 5 µM concentration of the dye for 10 min. Di-8-ANEPPS was excited at 514 nm and the fluorescence emission was monitored at wavelengths of >525 nm. To follow the rapid membrane potential changes with sufficient temporal resolution, we used the line-scan mode of the confocal microscope (250 ns/line).
Electroporation
Electroporation was performed using a homemade system capable of
generating mono- and bipolar square electrical pulses, with amplitudes
ranging from 0 to 2000 V and a duration of 1 to 1000 µs. The
amplitude and the waveform of the electric field exposure were
monitored using a digital storage oscilloscope (Tektronix, Beaverton,
Oreg.). The electroporation system essentially consists of a Perspex
chamber with two electrodes connected to a high-frequency voltage
source. The volume of the rectangular chamber (50 µl) was kept as
small as possible to enable the application of a uniform electric field
simultaneously with a consistent rapid perfusion of EM. The cells used
for the experiments were located in the central part of the narrow (3
mm) chamber filled with EM that brings into contact two platinum
electrodes. Perfusion of cells in the electroporation chamber avoids
artifacts resulting from the generation of electrolysis by-products,
such as O2 and H2. The temperature of the
extracellular solution was unaffected by the electrical pulse (data not
shown).
For video imaging studies, square monopolar pulses of either 10 or 50 µs duration were used. For the confocal studies, a square bipolar pulse of 50 µs duration in both directions (100 µs total duration) was applied in order to increase the chances of finding responsive cells at near-threshold levels of stimulation. For the confocal studies using di-8-ANEPPS, where changes in membrane potential were monitored, longer pulses of 1 ms were used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The stepwise quench of fura-2 by Mn2+ (Fig. 1C ) confirmed that the cation permeability of the cells increased with each electroporative pulse. Over the range of 0.6–1 kV/cm, both the quench of fura-2 by Mn2+ and the corresponding increases in [Ca2+]i showed a supralinear relationship to the field intensity (Fig. 2 A, B). These data indicate that increasing electroporative potentials cause greater cation entry and that at a field intensity of ~700 V/cm, a threshold was reached where [Ca2+]i responses were greatly enhanced (Fig. 2A ; filled squares). In cells where [Ca2+]i oscillations were initiated by a single electroporative pulse (e.g., Fig. 1B ), the Mn2+ quench of fura-2 revealed a single cation entry step simultaneous with the electroporation pulse, but no further discrete steps during the subsequent oscillations (data not shown).
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The supralinearity of the [Ca2+]i signals (Fig. 2A ) was not simply due to increasing Ca2+ influx at high field intensities, because the [Ca2+]i response displayed a > sixfold increase between 700 and 800 V/cm (Fig. 2A ), whereas the Mn2+ quench response increased by only twofold (Fig. 2B ). The enhancement of [Ca2+]i increases with field intensities above 700 V/cm was most likely due to amplification by Ca2+ release from intracellular stores, since this effect was absent during incubation of the cells in 20 mM caffeine to inhibit InsP3R function. Caffeine reduced the amplitude of the [Ca2+]i increases seen with larger (>700 V/cm) electroporation pulses and produced an almost linear relationship between field intensity and change in [Ca2+]i (Fig. 2A ; open circles). Removal of functional intracellular Ca2+ stores by prolonged treatment of cells with 200 nM thapsigargin substantially decreased electroporation-induced increases in [Ca2+]i (Fig. 2C ), also confirming that the major portion of the [Ca2+]i signals arose from Ca2+ release. Although HeLa cells express ryanodine receptors (RyRs) (27 , 28 ), these intracellular Ca2+ release channels did not seem to be involved in the electroporation-evoked [Ca2+]i signals, since preincubation of cells with 10 µM ryanodine for 30 min to block RyR opening did not alter the responses (data not shown).
As illustrated in Fig. 2A , low field intensities (500–700 V/cm) did not trigger large regenerative responses. An example of the low-amplitude, nonregenerative responses obtained with such low electroporative potentials is shown in Fig. 3 A. Repetitive application (1/min) of the electroporative pulse evoked only modest (~20 nM) increases in [Ca2+]i, which decayed back to basal levels in a matter of a few tens of seconds. But if such pulses were applied at a twofold higher frequency, they eventually triggered a regenerative response (Fig. 3B ). Similarly, low-frequency (1/min) electroporative pulses applied in the presence of a low (100 nM) histamine concentration also triggered a regenerative response (Fig. 3C ). This subthreshold histamine concentration was unable to evoke increases in [Ca2+]i when applied on its own for periods of up to 45 min (data not shown).
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A consistent feature of [Ca2+]i increases triggered by electroporation was that they displayed desensitization if large regenerative responses were induced, and the cells needed a period of recovery before similar responses could be elicited again. For example, although pulses of 1000 V/cm evoked consistent amplitude responses when applied at low frequency (1/6 min, Fig. 3D ), the [Ca2+]i increases triggered by the same stimulus rapidly desensitized when the frequency was increased to 1/min (Fig. 3E ). Since electroporation of cells invariably caused cation entry, as measured by Mn2+ quenching of fura-2 (e.g., Fig. 1C ), this desensitization did not reflect a progressive failure to electroporate the cells but rather the progressive failure of regenerative Ca2+ release.
These data suggest that electroporation caused an influx of Ca2+, which subsequently evoked regenerative Ca2+ release from InsP3Rs on intracellular stores. This temporal sequence of events is further supported by the observations that regenerative [Ca2+]i changes could occur with a latency of tens of seconds after the electroporative pulse (see below) and that electroporation-induced [Ca2+]i oscillations could persist for several minutes (Fig. 1B ).
Electroporation triggers regenerative
[Ca2+]i waves, but does not stimulate
InsP3 production
To confirm that electroporation triggered regenerative
Ca2+ release, we investigated the subcellular properties of
the electrically stimulated [Ca2+]i
transients using rapid confocal imaging. Application of suprathreshold
electric fields evoked [Ca2+]i signals that
began first in the regions of the cells closest to the electrodes, then
propagated throughout the cells in a nondecremental manner (Fig. 4
). Such regenerative [Ca2+]i waves could
be reproducibly elicited, with the initiation points consistently being
the parts of the cells most adjacent to the electrodes and with the
same direction of wave propagation. The velocities of these
electroporation-induced Ca2+ waves were only marginally
less than those evoked by histamine (Fig. 5
A, C; ref 4
). Preincubation of the cells with caffeine
(20 mM) markedly reduced the amplitude and velocity of such
[Ca2+]i waves to more slowly diffusing
signals that penetrated through the cytoplasm in a decremental manner
(Fig. 5B, C
).
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The observation that electroporation above a certain threshold led to a caffeine-inhibitable regenerative [Ca2+]i rise was surprising, since even though InsP3Rs are sensitive to Ca2+, they usually require coactivation with InsP3. We therefore tested whether electroporation simply mimicked hormone action on HeLa cells by evoking the production of InsP3, thereby triggering Ca2+ release from intracellular stores. We used the phospholipase C (PLC) inhibitor, U-73122, to prevent production of InsP3 during an electroporative pulse. U-73122 inhibited histamine-evoked [Ca2+]i signals in a concentration-dependent manner (IC50=273 nM; Fig 6A ). The structural analog U-73343, which is a much less potent PLC inhibitor, did not affect histamine-evoked Ca2+ signals at concentrations of up to 10 µM (Fig. 6 A). Electroporation-induced [Ca2+]i signals were unaffected by U-73122 at concentrations that virtually abolished histamine responses (Fig. 6C ).
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Electroporation caused rapid membrane disruption, followed by
Ca2+ influx and activation of Ca2+ puffs
With low field intensities, the Ca2+ influx occurring
during the electroporative pulse was small and usually barely
detectable, and it was the subsequent regenerative Ca2+
signals that confirmed electroporation had actually taken place.
However, with higher field intensities (
750 V/cm), transient
increases in [Ca2+]i were occasionally
observed that were far too rapid to be due to regenerative
Ca2+ release from InsP3Rs. Such
[Ca2+]i signals represented the influx of
Ca2+ through the electroporation-induced membrane pores,
and thus provided a way of estimating the life-time of these pores. An
example of such a Ca2+ influx signal is illustrated in
Fig. 7
. Concomitant with the electroporative pulse, a
[Ca2+]i increase was observed at one pole of
the cell (Fig. 7A, B
). The influx signal (Fig. 7B
) rapidly reached a peak [Ca2+]i
of ~200 nM, and triggered a more slowly developing regenerative
[Ca2+]i increase throughout the cell (Fig. 7C
). The expanded time course of the influx signal (Fig. 7D
) and its time derivative (Fig. 7E
) revealed
that the [Ca2+]i increase resulting from
membrane disruption lasted for only ~1 s, with a rising phase of 
4
ms. Such a response can only arise via near-instantaneous
Ca2+ entry after membrane disruption, followed by rapid
membrane resealing. We therefore conclude that the lifetime of the
electroporation-induced membrane pores is in the range of ~1–2 ms.
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To more closely monitor the effect of the electric field on the plasma membrane and its subsequent breakdown, we used the rapid voltage-sensitive indicator di-8-ANEPPS. Upon application of an electroporative pulse, a sudden change in membrane potential occurred in the regions of the cells most closely opposed to the electrodes (Fig. 8 A). The change in membrane potential correlated with the position of the cell relative to the electrodes; the membranes facing the anode or cathode displayed hyperpolarization or depolarization, respectively (Fig. 8A ).
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Two significant observations can be gleaned from the measurement of the di-8-ANEPPS signals. First, the electroporation-induced voltage change peaked and then started to decline within 0.5 ms of the start of the pulse (Fig. 8B ), even though the intensity of the field was constant during the exposure, which lasted for 1 ms (data not shown). The recovery of the membrane potential during the application of the electroporative pulse indicated that the resistance of the membrane rapidly decreased, and consequently that pores had been formed. Second, the change in membrane potential can be repeatedly elicited at a frequency of up to 10 Hz (Fig. 8C ), indicating rapid recovery of the membrane resistance, i.e., that the membrane has resealed. This showed that electroporation with pulses of 10-fold longer duration than those used to evoke Ca2+ signals in this study did not impose long-term membrane disruptions.
In the majority of cases, where electric fields of modest intensities
were used, [Ca2+]i increases arising directly
from Ca2+ entry were difficult to detect. Instead, there
was usually a variable latency of up to a few seconds between the
electroporative pulse and the onset of a
[Ca2+]i signal, suggesting that the rapid
undetectable Ca2+ influx had activated Ca2+
release (Fig. 9
Aa). Although the exact nature of the
[Ca2+]i signal triggered by electroporation
varied with the magnitude of the electroporative pulse, the most common
response to low field intensities (750 V/cm) was the activation of
Ca2+ puffs.
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We earlier determined the nature of hormonally induced Ca2+ puffs in HeLa cells (4 , 13 ), and found them to be rapid (rise time, <100 ms) and highly localized (full width at half maximum; FWHM up to 6 µm). Electroporation evoked similar elementary events, usually occurring in the regions of the plasma membrane most closely adjacent to the electrodes. These electroporation-induced Ca2+ puffs temporally and spatially resembled those induced by a hormone; they were visualized as spatially confined [Ca2+]i increases, with an FWHM of 2–7 µm (Fig. 9Ab ). A single Ca2+ puff was the most common response to an individual electroporative pulse, although trains of Ca2+ puffs were sometime evoked (Fig. 9B ).
The transition between nonregenerative and regenerative responses
was dependent on electroporation intensity and frequency
Although the electroporation-induced Ca2+ puffs
originated beneath the regions of the plasma membrane most closely
opposed to the electrodes, their exact location was unpredictable. In
addition, the threshold field intensity required to evoke
Ca2+ puffs differed both between cells and between various
parts of individual cells (data not shown). However, once a threshold
stimulus intensity had been found to elicit Ca2+ puffs,
pulsatile application of that voltage triggered repetitive
[Ca2+]i transients in the same cellular
location. The reproducible induction of Ca2+ puffs allowed
us to investigate the modes of recruitment of such elementary events
that induce globally regenerative [Ca2+]i
signals.
As illustrated in Fig. 2A , the intensity of the electroporation pulse applied to the cells determined the likelihood of triggering a regenerative [Ca2+]i signal. Subthreshold field intensities gave rise to discrete Ca2+ puffs, which at best triggered only a limited degree of local regeneration (Fig. 9B and Fig. 10 ). Despite repeated applications, such low-intensity fields did not trigger global [Ca2+]i signals. However, increasing the voltage applied to the cells could switch the response from spatially restricted Ca2+ signals to a regenerative [Ca2+]i wave (Fig. 10) .
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In addition to the effects of enhancing field intensity, increases in the frequency of electroporative pulses could also induce regenerative [Ca2+]i signals. The sequence of traces shown in Fig. 11 A–D illustrates the effects of increasing the frequency of a threshold electroporation pulse. At 0.05 or 0.1 Hz, the electroporative pulses evoked low-amplitude [Ca2+]i signals, which were restricted to only a couple of cellular regions (Fig. 11 A, B). Although there was some variability, presumably due to changes in the degree of local regenerativity, each electroporative pulse caused a localized [Ca2+]i increase. However, even after multiple pulses, a global [Ca2+]i signal was not triggered. At 0.2 Hz, spatially restricted [Ca2+]i signals were evoked, which were not able to recover back to basal levels before the onset of the next [Ca2+]i rise. Eventually, the elevated [Ca2+]i in the responsive regions diffused throughout the rest of the cell, causing a uniform [Ca2+]i elevation (Fig. 11C ), however, a regenerative [Ca2+]i signal was not triggered. Increasing the frequency of electroporation pulses to 0.5 Hz initially triggered discrete Ca2+ puffs and eventually led to a regenerative globally propagating [Ca2+]i wave (Fig. 11D ).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In HeLa cells, electroporation caused a Ca2+o-dependent [Ca2+]i increase (Fig. 1) that was proportional to the intensity of the electric field applied to the cells (Figs. 2 and 10) . Although the electroporation-evoked signals required Ca2+o, the [Ca2+]i increase appeared to arise largely through activation of regenerative Ca2+ release by intracellular Ca2+ channels, since it was substantially inhibited by caffeine (Fig. 2A and Fig. 5 ), required functional Ca2+ stores (Fig. 2C ), and could occur some time after the electroporative pulse (Fig. 4 and Fig. 9A ). In addition, whereas electroporation invariably caused Mn2+ quench of fura-2 (Fig. 1C ), [Ca2+]i increases were not always recorded (e.g., third electroporation pulse in Fig. 11A ). Furthermore, application of high-frequency electroporation pulses desensitized the [Ca2+]i response (Fig. 3) . Together, these observations suggest that a small, usually imperceptible Ca2+ influx activated regenerative Ca2+ release from internal stores.
Changes in the concentration of ions other than Ca2+—e.g., Na+ and K+—probably occurred during the formation of membrane pores by electroporation. Exact quantitation of changes in these ions was not undertaken during our study. However, we think it is reasonable to assume that the largest flux of ions will be Ca2+, as it has the largest electrochemical gradient across the cell. Since we are unable to measure the very small flux of Ca2+ during weak electroporation events, it is unlikely that we could measure changes in Na+ or K+. Furthermore, these ions are much more highly diffusible than Ca2+ in the cytoplasm, and therefore we would expect any changes in Na+ or K+ to be rapidly reequilibrated.
It was surprising to observe that electroporation on its own could trigger regenerative [Ca2+]i signals, since the activation of InsP3Rs is believed to require both InsP3 and Ca2+ 29-35) . However, the electroporation-evoked [Ca2+]i responses were not sensitive to the PLC inhibitor U-73122 (Fig. 6) , suggesting that it did not look to the cells as if they had been treated with an agonist. It seems that in the absence of hormonal stimulation, the basal intracellular InsP3 concentration is sufficient to allow regenerativity from InsP3Rs, providing a sufficient trigger Ca2+ is given. The effect of a subthreshold histamine concentration in sensitizing the cells to low-intensity electroporative pulses (Fig. 3) is consistent with the notion that electroporation-induced Ca2+ influx and modest intracellular InsP3 concentrations can elicit a synergistic effect.
Electroporation of HeLa cells with low (<700 V/cm) field intensities frequently generated Ca2+ puffs with a similar spatial spreading and time course to hormone-evoked elementary events (Fig. 9) . The ability to evoke such localized Ca2+ signals essentially results from the fact that electroporation is an asymmetric process that disrupts the membrane regions most closely opposed to the electrodes, particularly the membrane portion facing the anode. At low field intensities, only a small area of the plasma membrane may be disrupted, giving a highly localized Ca2+ influx. We suggest that this small Ca2+ influx triggers Ca2+ release from adjacent InsP3R, thus producing the observed Ca2+ puffs.
It should be noted, however, that although Ca2+ puffs were commonly observed, not all electroporation-evoked Ca2+ signals resembled hormonal Ca2+ puffs. Application of high-intensity voltages (>750 V/cm) sometimes caused subplasmalemmal [Ca2+]i rises that were too fast to be accounted for by regenerative Ca2+ release (Fig. 5) . These rapid signals most likely arose due to Ca2+ entry through the electroporation-induced membrane pores. The spatial spread of these signals was also distinct from Ca2+ puffs, with a semicircular diffusion shell immediately beneath the membrane (data not shown).
Hormone-evoked regenerative [Ca2+]i waves in HeLa cells result from the spatio-temporal recruitment of elementary Ca2+ events (4 , 13 ). In a previous study, we suggested that the transition from localized elementary events to global Ca2+ waves is determined by a change in the activity of the elementary signals themselves. Increases in frequency and/or amplitude of the Ca2+ puffs are required to evoke regenerative Ca2+ signals (13) . Elementary Ca2+ signals that do not advance in frequency or amplitude fail to trigger global signals. Cytoplasmic integration of the Ca2+ released during each puff is crucial to allow the ambient level of Ca2+ to reach the threshold for regenerativity to occur. With subthreshold levels of activity, the Ca2+ signals arising from individual Ca2+ puffs have a relatively short cytoplasmic lifetime before being buffered or sequestered. With sufficient activity, the Ca2+ released during each event overwhelms the cellular buffering mechanisms and is integrated by the cytoplasm to cause a progressive [Ca2+]i increase until the threshold for regnerativity is reached. The reproducibility of electroporation-induced Ca2+ signals allowed us to overcome the stochastic nature of hormone-evoked Ca2+ puffs and directly test this scheme.
Increasing the frequency at which low-intensity electroporative pulses were applied enhanced their ability to trigger regenerative Ca2+ waves (Fig. 11) . In addition, the effectiveness of single electroporative pulses in evoking Ca2+ waves was greater with higher voltages (Fig. 2A and Fig. 10 ). These data suggest that the electroporation-induced Ca2+ signals functionally mimic hormone-evoked Ca2+ puffs in generating regenerative Ca2+ waves when they provide a suitable trigger.
One significant difference between electroporation-induced events and hormonally stimulated elementary Ca2+ signals is that whereas the former are evoked beneath the plasma membrane in the periphery of the cells (Figs. 9 and 11) , the latter most frequently occur adjacent to the nucleus (36) . The reason why agonist-stimulated Ca2+ puffs are observed largely around the nucleus is unclear. However, the consequence is that a majority of Ca2+ waves are initiated from perinuclear regions. In contrast, electroporation-induced Ca2+ waves were initiated far from the nucleus (Fig. 5) . These data revealed that different parts of the cytoplasm can be functionally equivalent, and that the location of elementary signals determine initiation sites of regenerative responses.
The qualitative effect of triggering regenerative Ca2+ waves with particular electroporative frequencies or field intensities was observed whether the electroporation triggered Ca2+ puffs or simply a direct rapid Ca2+ entry signal (see above). This suggests that the important criterion for causing regenerative Ca2+ release is not the location or nature of the activating signal, but simply that a triggering [Ca2+]i threshold has to be attained.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Department of Physiology, University
College London, Gower Street, London, WC1E 6BT.
3 Abbreviations: [Ca2+]i, intracellular Ca2+; EM, external medium; InsP3, inositol 1,4,5-trisphosphate; InsP3R, InsP3 receptor; PLC, phospholipase C; RyR, ryanodine receptor.
Received for publication June 9, 1998.
Revision received October 19, 1998.
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F., Miklavi D.. Sensitivity of transmembrane voltage induced by applied electric fields—a theoretical analysis. Bioelectrochem. Bioenerg. 1997;43:285-291.
F.. Electroporationa method for introduction of non-permeable molecular probes. Mason W. T. eds. Fluorescent and Luminescent Probes 2nd Ed 1998 Academic Press Ltd London. In press.
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